組合蛋白(integrins)是一群位於細胞表面上的膜蛋白，它們的功能參與細胞之間的粘合、細胞的遷移以及訊息的傳遞等等。而組合蛋白牽涉到許多疾病的發生，所以它們也被視為具有潛力發展藥物設計的目標物。有些細胞之間的結合是藉由RGD(Arg-Gly-Asp)的序列，像細胞外間質細胞、血液細胞等，而且有許多的組合蛋白都可辨認RGD的序列。也有很多研究發現，在蛇毒中的去組合蛋白(disintegrins)其RGD周圍的胺基酸序列會去影響到它們和組合蛋白結合的特異性。Rhodostomin (Rho)是個有效抑制組合蛋白的去組合蛋白，在胺基酸48-53的位置是PRGDMP的序列。藉由細胞粘合分析的結果，我們發現ARGDGP和PRGDGP這兩個突變株會選擇性去抑制組合蛋白<font face="symbol">a</font>v<font face="symbol">b</font>3，另外，在RGD周圍序列是ARGDXP的突變株其對於組合蛋白<font face="symbol">a</font>5<font face="symbol">b</font>1有比較好的抑制效果。為了要探討組合蛋白<font face="symbol">a</font>v<font face="symbol">b</font>3和<font face="symbol">a</font>5<font face="symbol">b</font>1在辨認不同序列的結構決定要素，我們利用核磁共振光譜(NMR)的方法，決定出這些對於組合蛋白<font face="symbol">a</font>v<font face="symbol">b</font>3和<font face="symbol">a</font>5<font face="symbol">b</font>1特異性結合突變株的3D立體結構(ARGDMP、ARGDNP、ARGDGP和PRGDGP)。由我們結構上的分析，野生株PRGDMP和這些突變株的3D立體結構是一樣的;然而在ARGDGP和PRGDGP突變株中，RGD loop骨架的構形以及D51胺基酸側鏈的位向和野生株是不同的，它們的結構和我們之前發現一個對於組合蛋白<font face="symbol">a</font>v<font face="symbol">b</font>3特異性結合的突變株ARGDDL是相似的，所以我們認為在RGD loop構形上的不同，對於會選擇性去抑制組合蛋白<font face="symbol">a</font>v<font face="symbol">b</font>3是扮演一個重要的角色。另外由PRGDMP和ARGDMP的Model free動力學分析，發現把胺基酸P48突變成A48時，胺基酸R49的次序參數(S²)值是比較小的，這是源自於R₂值降低的結果，而含ARGDXP序列的突變蛋白對於組合蛋白<font face="symbol">a</font>5<font face="symbol">b</font>1有較好的抑制效果，在動力學性質上的不同似乎是一個重要的因素。還有我們也利用同源模擬法(homology modeling)，以組合蛋白<font face="symbol">a</font>v<font face="symbol">b</font>3的3D立體結構當模板(template)，製作出組合蛋白<font face="symbol">a</font>IIb<font face="symbol">b</font>3和<font face="symbol">a</font>5<font face="symbol">b</font>1的模型結構，而將對於不同組合蛋白<font face="symbol">a</font>IIb<font face="symbol">b</font>3、<font face="symbol">a</font>v<font face="symbol">b</font>3以及<font face="symbol">a</font>5<font face="symbol">b</font>1特異性結合的Rho突變株分別對接(docking)到組合蛋白中，進而想去鑑定出它們之間可能互相作用的胺基酸。由我們研究的結果可知道，去組合蛋白其RGD周圍的序列會去影響到它們的結構、動力學性質和功能，這樣的結果可幫助我們去設計有效抑制組合蛋白<font face="symbol">a</font>IIb<font face="symbol">b</font>3、<font face="symbol">a</font>v<font face="symbol">b</font>3以及<font face="symbol">a</font>5<font face="symbol">b</font>1的抑制物。

　　Integrins are a family of cell surface receptors that mediate cell cellular adhesion, cell migration and signal transduction. It is known that integrins play important roles in the initiation and progression of many common diseases. Therefore, antagonism of integrins provides an approach for the treatment and prevention of these diseases. The RGD sequence is the cell attachment site of a large number of adhesive ECM, blood, and cell surface proteins and nearly half of 24 known integrins recognize this sequence in their adhesive ligands. Many studies have showed that the amino acid residues flanking the RGD motif of snake venom disintegrins control their binding specificity. Rhodostomin (Rho) is a potent integrin inhibitor and contains a PRGDMP sequence at positions of 48-53. Based on our results of cell adhesion analyses, we found that Rho mutants containing either ARGDGP or PRGDGP sequence can selectively inhibit integrin <font face="symbol">a</font>v<font face="symbol">b</font>3 and Rho mutants containing ARGDXP sequence in the RGD loop have better inhibitory activity to integrin <font face="symbol">a</font>5<font face="symbol">b</font>1. To understand structural determinants of integrins <font face="symbol">a</font>v<font face="symbol">b</font>3 and <font face="symbol">a</font>5<font face="symbol">b</font>1 recognition, we have determined 3D structures of these mutant proteins (ARGDMP, ARGDNP, ARGDGP and PRGDGP) by using NMR spectroscopy. Based on our structural analyses, the tertiary folds of wild-type and mutant proteins are the same. However, the backbone conformation of the RGD loop and the side-chain orientation of the D51 residue of mutant proteins (ARGDGP and PRGDGP) differ from those of wild-type Rho. Their structures are similar to our reported structure of the ARGDDL mutant, an integrin <font face="symbol">a</font>v<font face="symbol">b</font>3-specific protein. These results suggest that these conformational differences may be responsible for their inhibitory selectivity to integrin <font face="symbol">a</font>v<font face="symbol">b</font>3. In addition, model-free analyses of PRGDMP and ARGDMP proteins reveal that mutation of P48 to A caused a decrease in the order parameter (S²) of the R49 residue of the ARGDMP protein, mainly due to the decrease of R₂. This difference may be responsible for the better inhibitory activity of Rho mutants containing ARGDXP sequence to integrin <font face="symbol">a</font>5<font face="symbol">b</font>1. Using 3D structure of <font face="symbol">a</font>v<font face="symbol">b</font>3 integrin as template, 3D model structures of integrins <font face="symbol">a</font>IIb<font face="symbol">b</font>3 and <font face="symbol">a</font>5<font face="symbol">b</font>1 were generated by using homology modeling. Docking integrins <font face="symbol">a</font>IIb<font face="symbol">b</font>3, <font face="symbol">a</font>v<font face="symbol">b</font>3, and <font face="symbol">a</font>5<font face="symbol">b</font>1-specific Rho mutants to their receptors will be used to identify their possible interacting amino acids. Our findings suggest that the residues flanking the RGD motif of disintegrins regulate their structure, dynamics and function. These results will be used to design potent integrins <font face="symbol">a</font>IIb<font face="symbol">b</font>3, <font face="symbol">a</font>v<font face="symbol">b</font>3, and <font face="symbol">a</font>5<font face="symbol">b</font>1-specific antagonists.

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